Inhibitor of transforming growth factor β and A method of inhibiting the biological effects of transforming growth factor

ABSTRACT

A method of inhibiting, ameliorating or reversing the effects of TGF-β in biological systems by exposing the bioligical systems with an agent which substantially represents active site peptide of the TGF-β molecules.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority from an earlier filed provisionalpatent application Ser. No. 60/050,202, filed Jun. 19, 1997.

ACKNOWLEDGMENT

This invention was made with the US Government support awarded by theNational Institutes of Health. The US Government may have certain rightsin the invention.

BACKGROUND OF THE INVENTION

Transforming growth factor β (TGF-β) is a family of 25-kDa structurallyhomologous dimeric proteins containing one interchain disulfide bond andfour intrachain disulfide bonds. The TGF-β family is composed ofthree-known members (TGF-β₁, TGF-β₂, and TGF-β₃) in mammalian species.TGF-β is a bifunctional growth regulator: it is a growth inhibitor forepithelial cells, endothelial cells, T-cells, and other cell types and amitogen for mesenchymal cells. TGF-β also has other biologicalactivities, including stimulation of collagen, fibronectin, andplasminogen activator inhibitor 1 (PAI-1) synthesis, stimulation ofangiogenesis, and induction of differentiation in several cell lineages.

TGF-β has been implicated in the pathogenesis of various diseases suchas intimal hyperplasia following angioplasty, tissue fibrosis, andglomerulonephritis. Neutralizing antibodies to TGF-β have been usedexperimentally to reduce scarring of wounds, to prevent lung injury inadult respiratory distress syndrome (ARDS), and to block restenosisfollowing angioplasty in animal models. These promising results warrantthe development of TGF-β antagonists (inhibitor) that might be useful ininhibiting, ameliorating or reversing the effects of TGF-β and treatingdiseases.

SUMMARY OF THE INVENTION

A method of inhibiting, ameliorating or reversing the effects of TGF-βin biological systems, comprising the step of exposing said biologicalsystems with a binding agent, said binding agent is a peptide whichsubstantially resembles a segment of the TGF-β molecules.

The binding agent is a peptide which substantially resembles an activesite of the TGF-β molecules, hereinafter referred to as active sitepeptide.

The binding agent may be in the form of a peptide or other chemicalagents which substantially resembles an active site of the TGF-βmolecule, the binding agents occupy the TGF-β cellular receptors makingthe TGF-β cellular receptors unavailable for the binding of TGF-βmolecules.

The biological systems may be either in-vitro or in-vivo.

Most preferably, the binding agent of TGF-β substantially corresponds toa peptide having an amino acid sequence substantially extending fromresidue 41 to residue 65 of TGF-β amino acid sequence. Most preferably,said binding agents correspond to an amino acid sequence motifpreferably represented by WSXD (SEQ ID NO:10) and/or RSXD (SEQ IDNO:11), wherein X represents any amino acid.

Yet another object of the present invention is to provide an active siteof TGF-β molecules. And to provide an amino acid sequence whichsubstantially represents an active site of TGF-β molecules.

Yet another object of the present invention is to provide an agonist ofTGF-β molecules, comprising a carrier molecule have a plurality of saidbinding agent. Furthermore, a method of producing an agonist of TGF-βmolecules, comprising the step of conjugating a plurality of saidbinding agents to a carrier molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Effect of various concentrations of pentacosapeptides,decapeptides, and their structural variants on ¹²⁵I-TGF-β₁, (Panels Aand D), ¹²⁵I-TGF-β₂ (Panel B), and ¹²⁵I-TGF-β₃ (Panel C) binding toTGF-β receptors in mink lung epithelial cells.

Cells were incubated with ¹²⁵I-TGF-β₁ (Panels A and D), ¹²⁵I-TGF-β₂(Panel B), and ¹²⁵I-TGF-β₃ (Panel C) both with and without 100-foldexcess of unlabeled TGF-β isoforms and various concentrations of β₁ ²⁵(41-65)(SEQ ID NO:4), β₂ ²⁵ (41-65)(SEQ ID NO:5), and β₃ ²⁵ (41-65)(SEQID NO:6)(Panels A, B, and C) or of β₁ ¹⁰ (49-58)(SEQ ID NO:7), β₂ ¹⁰(49-58)(SEQ ID NO:8), β₃ ¹⁰ (49-58)(SEQ ID NO:9), β₁ ¹⁰ (49-58) W52A, β₂¹⁰ (49-58) S53A, β₂ ¹⁰ (49-58) D55A, β₁ ²⁵ (41-65) W52A/D55A and β₃ ²⁵(41-65) R52A/D55A (Panel D). The specific binding of ¹²⁵I-labeled TGF-βisoforms was then determined. The specific binding obtained in theabsence of peptide antagonists was taken as 0% inhibition. The specificbinding (0% inhibition) of ¹²⁵I-TGF-β₁, ¹²⁵I-TGF-β₂, and ¹²⁵I-TGF-β₃were 3930+540 cpm/well, 4512±135 cpm/well and 4219±125 cpm/well,respectively. The error bars are means ± of triplicate cultures. FIG. 2.¹²⁵I-TGF-β₁-affinity labeling of cell-surface TGF-β receptors afterincubation of mink lung epithelial cells with ¹²⁵I-TGF-β₁ in thepresence of various concentrations of β₁ ²⁵ (41-65) and β₃ ²⁵ (41-65).

Cells were incubated with ¹²⁵I-TGF-β₁ in the presence of 100-fold excessof unlabeled TGF-β₁ (lane 1) and of various concentrations of β₁ ²⁵(41-65) (lanes 8-13) and β₃ ²⁵ (41-65) (lanes 2-7). The¹²⁵I-TGF-β₁-affinity labeling was carried out in the presence of DSS.The ¹²⁵I-TGF-β₁ affinity-labeled TGF-β receptors were analyzed by 5%SDS-polyacrylamide gel electrophoresis and autoradiography. The arrowindicates the location of the ¹²⁵I-TGF-β₁ affinity-labeled type V TGF-βreceptor (TβR-V). The brackets indicate the locations of the ¹²⁵I-TGF-β₁affinity-labeled type I, type II, and type III TGF-β receptors (TβR-I,TβR-II, and TβR-III).

FIG. 3. Effect of β₁ ²⁵ (41-65) on TGF-β₁-induced growth inhibition asmeasured by DNA synthesis (Panels A and B) and TGF-β₁-induced PAI-1expression (Panel C) in mink lung epithelial cells.

(Panel A) Cells were incubated with various concentrations of TGF-β₁ inthe presence of 18 μM β₁ ²⁵ (41-65). [Methyl-³H]thymidine incorporationinto cellular DNA was then determined. The [methyl-³H]thymidineincorporation into cellular DNA in cells treated with and without 10 pMTGF-β₁ were taken as 100 and 0% inhibition. The error bars are means±S.D. of triplicate cultures. (Panel B) Cells were incubated with 0.25 pMTGF-β, in the presence of various concentrations of β₁ ²⁵ (41-65). The[methyl-³H]thymidine incorporation into cellular DNA in cells treatedwith and without 10 pM TGF-β₁ were taken as 100 and 0% inhibition,respectively. The error bars are means± S.D. of triplicate cultures.(Panel C) Cells were treated with 0.25 and 2.5 pM TGF-β₁ and variousconcentrations of β₁ ²⁵ (41-65) for 3 hr. The transcriptionalexpressions of PAI-1 and glyceraldehyde-3-phosphate dehydrogenase(G3PDH) were determined by Northern blot analysis.

FIG. 4. Effect of β₁ ²⁵ (41-65)-CA and β₁ ²⁵ (41-65)-BSA conjugates on¹²⁵I-TGF-β₁ binding to TGF-β receptors in mink lung epithelial cells(Panel A) and on mink lung epithelial cell growth as measured by DNAsynthesis (Panel B).

(Panel A) Cells were incubated with ¹²⁵I-TGF-β₁ in the presence andabsence of 100-fold excess of unlabeled TGF-β₁ and variousconcentrations of β₁ ²⁵ (41-65)-CA conjugate. The specific binding of¹²⁵I-TGF-β₁ was then determined. The specific binding of ¹²⁵I-TGF-β₁obtained in the absence of the conjugates was taken as 0% inhibition.The error bars are means± S.D. of triplicate cultures. (Panel B) Cellswere treated with various concentrations of β₁ ²⁵ (41-65)-CA or β₁ ²⁵(41-65)-BSA conjugate. [Methyl-³H]thymidine incorporation into cellularDNA was determined. The [methyl-³H]thymidine incorporation into cellularDNA in cell treated with and without 10 pM TGF-β₁ were taken as 100 and0% inhibition, respectively. The error bars are means± S.D. oftriplicate cultures.

FIG. 5A shows the amino acid sequence of human TGF-β₁ (SEQ ID NO:1),human TGF-β₂ (SEQ ID NO:2), and human TGF-β₃ (SEQ ID NO:3); FIG. 5Bshows the amino acid sequence of β₁ ²⁵(41-65)(SEQ ID NO:4), β₂²⁵(41-65)(SEQ ID NO:5), and β₃ ²⁵(41-25)(SEQ ID NO:6), which consist ofamino acids 41-65 of TGF-β₁, TGF-β₂, and TGF-β₃, respectively.

DESCRIPTION OF THE INVENTION

The object of the invention is to develop TGF-β antagonists orinhibitors with specificities toward the type V TGF-β receptor and otherTGF-β receptor types (type I, type II, and type III receptors). It wasdiscovered that three chemically synthesized pentacosapeptides (i.e.binding agents), β₁ ²⁵ (41-65), β₂ ²⁵ (41-65), and β₃ ²⁵ (41-65), whoseamino acid sequences were derived from and correspond to the 41st to65th amino acid residues of TGF-β₁, TGF-β₂, and TGF-β₃, inhibit thebinding of radiolabeled TGF-β₁, TGF-β₂, and TGF-β₃ to TGF-β receptors inmink lung epithelial cells (i.e. cellular receptors). It was alsodiscovered that the W/RXXD motif in the sequences determines theirpotencies and that they block TGF-β-induced growth inhibition andTGF-β-induced expression of PAI-1 in mink lung epithelial cells. It wasalso discovered that these TGF-β peptide antagonists can be converted topartial agonists (i.e. agent which mimics the effects of TGF-β) byconjugation to carriers such as proteins or synthetic polymers.

To develop synthetic peptide antagonists of TGF-β, sevenpentacosapeptides were synthesized β₁ ²⁵ (21-45), β₁ ²⁵ (41-65), β₁ ²⁵(51-75), β₁ ²⁵ (61-85), β₁ ²⁵ (71-95), and β₁ ²⁵ (81-105), whose aminoacid sequences overlap one another and cover most of the human TGF-β₁molecule, the monomer of which has 112 amino acid residues (1). Theantagonist activities of these peptides were first tested for theirabilities to inhibit ¹²⁵I-labeled TGF-β₁ (¹²⁵I-TGF-β₁) binding tocell-surface TGF-β receptors in mink lung epithelial cells, a standardmodel system for investigating TGF-β receptor types and TGF-β-inducedcellular responses (2). β₁ ²⁵ (41-65), whose amino acid sequencecorresponds to the 41st-65th amino acid residues of TGF-β₁, completelyinhibited the ¹²⁵I-TGF-β₁ binding (specific binding withoutpeptides=3672±524 cpm/well) to TGF-β receptors in mink lung epithelialcells at 34 μM. The other six pentacosapeptides did not show any effecton ¹²⁵I-TGF-β₁ binding to TGF-β receptors in these epithelial cells evenat a concentration of 136 μM. This suggests that β₁ ²⁵ (41-65) is aTGF-β inhibitor or antagonist and contain an active-site amino acidsequence of TGF-β₁.

TGF-β isoforms (TGF-β₁, TGF-β₂, and TGF-β₃) have been shown to exhibitdifferent potencies in inducing cellular responses in certain cell typesor systems. There is 70% amino acid sequence homology at the 41st to65th amino acid residues among these three TGF-β isoforms (1-3). Todetermine the potencies of β₁ ²⁵ (41-65), β₂ ²⁵ (41-65), and β₃ ²⁵(41-65) in terms of TGF-β antagonist activity, applicant measured theeffects of these peptides on the binding of ¹²⁵I-labeled TGF-β₁, TGF-β₂,and TGF-β₃ to TGF-β receptors in mink lung epithelial cells. As shown inFIG. 1, both β₁ ²⁵ (41-65) and β₂ ²⁵ (41-65) inhibited ¹²⁵I-TGF-β₁ and¹²⁵I-TGF-β₂ binding to TGF-β receptors in a concentration-dependentmanner with an IC₅₀ of ˜1-2 μM (FIG. 1, A and B). β₃ ²⁵ (41-65) wasweaker with an IC₅₀ of ˜20 μM for inhibiting ¹²⁵I-TGF-β₁ and ¹²⁵I-TGF-β₂binding to TGF-β receptors (FIG. 1A and B). In contrast, β₁ ²⁵ (41-65)and β₃ ²⁵ (41-65) showed equal potency (IC₅₀=˜0.06-0.08 μM) when¹²⁵I-TGF-β₃ was used as ligand for testing the inhibitory activity (FIG.1C). β₂ ²⁵ (41-65) also had an IC₅₀ of ˜0.08 μM for inhibiting¹²⁵I-TGF-β₃ binding to TGF-β receptors in these epithelial cells (datanot shown). These results show that both β₁ ²⁵ (41-65) and β₂ ²⁵ (41-65)are more potent antagonists than β₃ ²⁵ (41-65) for ¹²⁵I-TGF-β₁ and¹²⁵I-TGF-β₂, and that all three pentacosapeptides are potent antagonistsfor ¹²⁵I-TGF-β₃ with equal IC₅₀.

The region spanning residues 41-65 includes a loop in thethree-dimensional structures of TGF-β₁ and TGF-β₂ (4,5). This loop isaccessible to solvent according to X-ray and NMR analyses (4,5). Thereare two reasons why a WSXD (for TGF-β₁ and TGF-β₂) or RSXD (for TGF-β₃)motif in the loop is a good candidate site whereby thesepentacosapeptides and their parent molecules could interact with TGF-βreceptors. The W/RSXD (52nd-55th amino acid residues) motif is locatedon the exposed surface of the loop, and the side chains of the aminoacid residues in the motif orient toward the solvent (4,5). Also, thismotif may determine the affinities of β₁ ²⁵ (41-65), β₂ ²⁵ (41-65), andβ₃ ²⁵ (41-65), and their parent molecules for binding to TGF-βreceptors. Both β₁ ²⁵ (41-65) and β₂ ²⁵ (41-65) share the same motif(WSXD) and have equal potencies (IC₅₀=1-2 μM) for the inhibition of¹²⁵I-TGF-β₁ binding to TGF-β receptors. β₃ ²⁵ (41-65) possesses adistinct motif of RSXD and is a weaker inhibitor (IC₅₀ of 20 EM). TheKds for TGF-β₁ and TGF-β₂ binding to the type V TGF-β receptor areidentical (˜0.4 nM), whereas the Kd of TGF-β₃ binding to the type Vreceptor is higher (˜5 nM) (6). To test the possibility that the W/RSXDmotif is the active site of these peptides, applicant synthesized threedecapeptides designated β₁ ¹⁰ (49-58), β₂ ¹⁰ (49-58), and β₁ ¹⁰ (49-58)whose amino acid sequences correspond to the 49th-58th amino acidresidues of TGF-β₁, TGF-β₂, and TGF-β₃, respectively. The W/RSXDvariants of these decapeptides in which the W-52, S-53, or D-55 residuewas replaced by an alanine residue were also synthesized and designatedβ₂ ¹⁰ (49-58) W52A, β₂ ¹⁰ (49-58) S53A, and β₂ ¹⁰ (49-58) D55A,respectively. The abilities of these decapeptides to inhibit ¹²⁵I-TGF-β₁binding to TGF-β receptors in mink lung epithelial cells were thenexamined. As shown in FIG. 1D, both β₁ ¹⁰ (49-58) and β₂ ¹⁰ (49-58)inhibited the ¹²⁵I-TGF-β₁ binding to TGF-β receptors in aconcentration-dependent manner with an IC₅₀ of ˜40-70 μM. β₃ ³¹ (49-58)did not show any inhibitory activity at concentrations up to 300 μM. β₂¹⁰ (49-58) S53A was equipotent with an IC₅₀ of 40 μM. The othervariants, β₂ ¹⁰ (49-58) W52A and β₂ ¹⁰ (49-58) D55A, failed to inhibit¹²⁵I-TGF-β₁ binding to TGF-β receptors in these epithelial cells.Identical experiments with β₁ ¹⁰ (49-58) W52A, β₁ ¹⁰ (49-58) S53A, andβ₁ ¹⁰ (49-58) D55A were also carried out, and the results were similarto those shown in FIG. 2D with the β₂ ¹⁰ (49-58) variants (data notshown). These results suggest that the WXXD motif is important for theinhibitory activity ofthe decapeptides β₁ ¹⁰ (49-58) and β₂ ¹⁰ (49-58).To prove that the W/RXXD motif is also important for the inhibitoryactivities of the pentacosapeptides β₁ ²⁵ (41-65) and B₃ ²⁵ (41-65),applicant prepared variants of β₁ ²⁵ (41-65) and β₃ ²⁵ (41-65), in whichboth W- or R-52 and D-55 were replaced by alanine residues. These weredesignated β₁ ²⁵ (41-65) W52A/D55A and β₃ ²⁵ (41-65) R52A/D55A,respectively, and tested for their inhibitory activities. FIG. 1D showsthat β₁ ²⁵ (41-65) W52A/D55A and β₃ ²⁵ (41-65) R52A/D55A did not inhibit¹²⁵I-TGF-β₁ binding to TGF-β receptors. These results support that themotif W/RXXD is involved in the interactions of the peptide antagonistseith TGF-β receptors.

Mink lung epithelial cells express all known and well-characterizedTGF-β receptors (type I, type II, type III, and type V receptors) (6).To determine the relative sensitivities of TGF-β receptor types toinhibition by β₁ ²⁵ (41-65) and β₃ ²⁵ (41-65) with respect to ligandbinding, applicant performed ¹²⁵I-TGF-β₁-affinity labeling ofcell-surface TGF-β receptors after incubation of mink lung epithelialcells with ¹²⁵I-TGF-β₁ in the presence of various concentrations of β₁²⁵ (41-65) and β₃ ²⁵ (41-65). As shown in FIG. 2, all cell-surface TGF-βreceptors (type I, type II, type III, and type V receptors) wereaffinity-labeled with ¹²⁵I-TGF-β₁ in the absence of the antagonists(lanes 7 and 13). β₁ ²⁵ (41-65) appeared to inhibit the¹²⁵I-TGF-β₁-affinity labeling of all TGF-β receptor types in aconcentration-dependent manner (lanes 8-12). However, B₁ ²⁵ (41-65)inhibition of the ¹²⁵I-TGF-β₁-affinity labeling of the type V TGF-βreceptor was greater than its inhibition of other TGF-β receptor types.The ¹²⁵I-TGF-β₁-affinity labeling of the type V TGF-β receptor wasalmost completely abolished by β₁ ²⁵ (41-65) at 2.3 μM , whereas the¹²⁵I-TGF-β₁-affinity labeling of other TGF-β receptor types was onlypartially inhibited (30-40%) (FIG. 2, lane 10). This result isconsistent with applicant's observation that the affinity for TGF-β₁binding to the type V TGF-β receptor is ˜20-40-fold lower than those forTGF-β₁ binding to other TGF-β receptor types (6). β₃ ²⁵ (41-65) showed aweak activity in blocking the ¹²⁵I-TGF-β₁-affinity labeling of the typeV TGF-β receptor (FIG. 2, lanes 2-5).

It has been demonstrated that β₁ ²⁵ (41-65), β₂ ²⁵ (41-65), and β₃ ²⁵(41-65) are potent inhibitors for ¹²⁵I-TGF-β₁ binding to TGF-βreceptors. To fulfill the criteria for TGF-β antagonists or inhibitor,these pentacosapeptides have to be shown to block TGF-β-induced cellularresponses. One prominent biological activity of TGF-β is growthinhibition. Applicant investigated the effect of β₁ ²⁵ (41-65) onTGF-β₁-induced growth inhibition by exposing mink lung epithelial cellsto various concentrations of TGF-β₁ in the presence of 18 μM β₁ ²⁵(41-65) and measuring cellular DNA synthesis. As shown in FIG. 3A, DNAsynthesis inhibition induced by 0.025 and 0.25 pM TGF-β₁ was completelyblocked by β₁ ²⁵ (41-55). In the presence of β₁ ²⁵ (41-65), thedose-response curve of TGF-β₁ shifted to the right. β₁ ²⁵ (41-65)blocked TGF-β₁-induced growth inhibition in a concentration-dependentmanner (FIG. 3B). It is important to note that β₁ ²⁵ (41-65) (0.1 to 36μM) did not have an effect on DNA synthesis in the absence of TGF-β₁.These results suggest that β₁ ²⁵ (41-65) is a TGF-β antagonist whichblocks TGF-β-induced growth inhibition. The other prominent biologicalactivity of TGF-β is transcriptional activation of collagen,fibronectin, and PAI- 1. To see if β₁ ²⁵ (41-65) is able to block thisactivity, applicant investigated the effect of β₁ ²⁵ (41-65) on PAI-1expression in mink lung epithelial cells stimulated by 0.25 and 2.5 pMTGF-β₁. As shown in FIG. 3C, β₁ ²⁵ (41-65) completely blocked the PAI-1expression stimulated by TGF-β₁ (lane 7 versus lanes 3 and 5). Theseresults further support that β₁ ²⁵ (41-65) is a potent TGF-β antagonist.

The dimeric structure of TGF-β has been shown to be required for itsbiological activities. The hetero-oligomerization of TGF-β receptorsinduced by the TGF-β dimer appears to trigger signaling. If β₁ ²⁵(41-65) contains the active site sequence involved in the interaction ofTGF-β₁ with TGF-β receptors, one may be able to convert its antagonistactivity to agonist activity by conjugating β₁ ²⁵ (41-65) to carrierproteins, such that the β₁ ²⁵ (41-65)-protein conjugates would carrymultiple valences of the putative active site. To test this possibility,β₁ ²⁵ (41-65) was conjugated to carrier proteins CA (carbonic anhydrase)and BSA (bovine serum albumin) using the cross-linking agent DSS. DSSmainly cross-links the α-amino group of β₁ ²⁵ (41-65) to the ε-aminogroups of the carrier proteins. The β₁ ²⁵ (41-65)-BSA and β₁ ²⁵(41-65)-CA conjugates contained ˜5-10 molecules of β₁ ²⁵ (41-65) permolecule of carrier protein. As shown in FIG. 4A, the β₁ ²⁵ (41-65)-CAconjugate inhibited ¹²⁵I-TGF-β₁ binding to TGF-β receptors in mink lungepithelial cells with an IC₅₀ of ˜0.05 μM. The β₁ ²⁵ (41-65)-BSAconjugate had a similar IC₅₀ of ˜0.06 μM (data not shown). These IC₅₀are ˜20-fold lower than that of β₁ ²⁵ (41-65) prior to conjugation. Inthe control experiments, both BSA and CA conjugated without peptides didnot have inhibitory activity. These results suggest that the multiplevalences of the active site in the protein conjugates enhance itsaffinity for binding to TGF-β receptors. Potential agonist activities ofthe β₁ ²⁵ (41-65)-protein conjugates was also examined. As shown in FIG.4B, both β₁ ²⁵ (41-65)-CA and β₁ ²⁵ (41-65)-BSA conjugates induced asmall but significant growth inhibition as measured by DNA synthesiswith an ED₅₀ of 0.1 μM, although neither showed significant effects onthe expression of PAI-1 in mink lung epithelial cells (data not shown).The growth inhibition (30-40%) induced by 0.2 μM β₁ ²⁵ (41-65)-CA couldbe abolished in the presence of 10 μM β₁ ²⁵ (41-65) (data not shown).These results suggest that these β₁ ²⁵ (41-65)-protein conjugates arepartial TGF-β agonists.

Applicant shows that the W/RXXD motif is a primary site involved in theinteraction with TGF-β receptors. This is supported by several lines ofevidence including: 1) among seven pentacosapeptides whose amino acidsequences cover most of the TGF-β₁ molecule, only β₁ ²⁵ (41-65), whichcontains the W/RXXD motif in the middle of the peptide amino acidsequence, has TGF-β antagonist activity; 2) pentacosapeptides anddecapeptides containing this W/RXXD motif are potent TGF-β antagonists;3) replacement of W-52/R-52 and D-55 by alanine residues abolishes theantagonist activities of these decapeptides and pentacosapeptides; 4)conjugation of the β₁ ²⁵ (41-65) antagonist to carrier proteins createsa partial TGF-β agonist; 5) several proteins that possess W/RXXD motifshave TGF-β agonist and antagonist activities.

Most of the experiments in this study were performed using mink lungepithelial cells. However, the antagonist activities of thepentacosapeptides (i.e. binding agents or active site peptides) havebeen substantially reproduced using a human line i.e. human lungfibroblasts (data not shown). Since TGF-β is highly conserved across thespecies, this invention disclosed herein is substantially valid acrossspecies.

Experimental Procedures

Material

Na¹²⁵I (17 Ci/mg) and [methyl-³H]thymidine (67 Ci/mmole) were purchasedfrom ICN Radiochemicals (Irvine, Calif.). High molecular-weight proteinstandards (myosin, 205 kDa; β-galactosidase, 116 kDa; phosphorylase, 97kDa; bovine serum albumin, 66 kDa), chloramine T, bovine serum albumin(BSA), and human carbonic anhydrase I (CA) were purchased from SigmaCompany (St. Louis, Mo.). Disuccinimidyl suberate (DSS) was obtainedfrom Pierce (Rockford, IL). TGF-β₁ was purchased from AustralBiologicals (San Ramon, Calif.). TGF-β₂ and TGF-β₃ were purchased fromR&D Systems (Minneapolis, Minn.).

Preparation of Pentacosapeptides and Decapeptides.

The amino acid sequences of all pentacosapeptides and decapeptides werederived from those of TGF-β₁, TGF-β₂, and TGF-β₃. For pentacosapeptidesβ₁ ²⁵ (41-65), β₂ ²⁵ (41-65), and β₃ ²⁵ (41-65), other versions in whichcysteine-44 and cysteine-48 were replaced by serine residues were alsosynthesized. These C44S/C48S versions of β₁ ²⁵ (41-65) and β₂ ²⁵(41-65), and β₃ ²⁵ (41-65) had the same TGF-β antagonist activity. TheC44S/C48S versions had better stability in solution during storage, sothey were used in most of the experiments. The peptides were synthesizedusing tert-butoxycarbonyl chemistry on an Applied Biosystems Model 431Apeptide synthesizer and purified using Sephadex G-25 columnchromatography and reverse-phase HPLC (C-8 column). The purity of thesynthesized peptides were verified by automated Edman degradation on anApplied Biosystems Model 477A gas/liquid phase protein sequenator withan on-line Applied Biosystems Model 120A phenylthiohydantoin amino acidanalyzer. The purity of all peptides was estimated to be ≧95%.

Preparation of β₁ ²⁵ (41-65)-Carbonic Anhydrase (CA) and β₁ ²⁵(41-65)-Bovine Serum Albumin (BSA) Conjugates

One hundred fifty μl of 3 mM β₁ ²⁵ (41-65) in phosphate buffer saline(pH adjusted to 9.0) was mixed with 300 μl of 0.1 M NaHCO₃ (pH ˜9.0)containing BSA or CA (0.5 mg) and 10 μl of 27 mM DSS in dimethylsulfoxide. After 18 hr at 4° C., the reaction mixture was mixed with 50μl of 1 M ethanolamine HCl in 0.1 M NaHCO₃ (˜pH 9.0). After 2 hr at roomtemperature, the reaction mixture was dialyzed against 2 liters of 0.1 MNaHCO₃ (pH 9.0). After four changes of the dialysis solution, the samplewas stored at 4° C. prior to use. The molar ratio of β₁ ²⁵(41-65)/carrier protein in the conjugate was determined by amino acidcomposition analysis.

Specific Binding of ¹²⁵I-labeled TGF-β₁, TGF-β₂, and TGF-β₃(¹²⁵I-TGF-β₁, ¹²⁵I-TGF-β₂, and ¹²⁵I-TGF-β₃) to TGF-β Receptors in MinkLung Epithelial Cells

¹²⁵I-TGF-β₁, ¹²⁵I-TGF-β₂, and ¹²⁵I-TGF-β₃ were prepared by iodination ofTGF-β₁, TGF-β₂, and TGF-β₃ with Na¹²⁵I as described previously (7). Thespecific radioactivities of ¹²⁵I-TGF-β₁, ¹²⁵I-TGF-β₂, and ¹²⁵I-TGF-β₃were 1-3×10⁵ cpm/ng. Mink lung epithelial cells were grown on 24-wellclustered dishes to near confluence in Dulbecco's modified Eagle medium(DMEM) containing 10% fetal calf serum. The epithelial cells wereincubated with 0.1 nM ¹²⁵I-TGF-β₁, ¹²⁵I-TGF-β₂, or ¹²⁵I-TGF-β₃ both withand without 100-fold excess of unlabeled TGF-β₁, TGF-β₂, or TGF-β₃ inbinding buffer (7). After 2.5 hr at 0° C., the cells were washed twotimes with binding buffer, and the cell-associated radioactivity wasdetermined. The specific binding of ¹²⁵I-labeled TGF-β isoforms to TGF-βreceptors in the cells was calculated by subtracting non-specificbinding (in the presence of 100-fold excess of the unlabeled TGF-βisoforms) from total binding. All experiments were carried out intriplicate cell cultures.

¹²⁵I-TGF-β₁-affinity Labeling of Cell-surface TGF-β Receptors in MinkLung Epithelial Cells

Mink lung epithelial cells grown on 60-mm Petri dishes were incubatedwith 0.1 InM ¹²⁵I-TGF-β₁ in the presence of various concentrations ofP²⁵ (41-65) or β₃ ²⁵ (41-65) in binding buffer. After 2.5 hr at 0° C.,¹²⁵I-TGF-β₁-affinity labeling was carried out in the presence of DSS asdescribed previously. The ¹²⁵I-TGF-β₁ affinity-labeled TGF-β receptorswere analyzed by 5% SDS-polyacrylamide gel electrophoresis underreducing conditions and autoradiography.

[Methyl-³H]thymidine Incorporation

Mink lung epithelial cells grown on 24-well clustered dishes wereincubated with various concentrations of TGF-β₁ in the presence andabsence of β₁ ²⁵ (41-65) or with various concentrations of β₁ ²⁵(41-65)-CA, and β₁ ²⁵ (41-65)-BSA in DMEM containing 0.1% fetal calfserum. After 16 hr at 37° C., the cells were pulsed with 1 μCi/ml of[methyl-³H]thymidine for 4 hr. The cells were then washed twice with 1ml of 10% trichloroacetic acid and once with 0.5 ml of ethanol:ether(2:1, v/v). The cells were then dissolved in 0.4 ml of 0.2N NaOH andcounted with a liquid scintillation counter.

RNA Analysis

Mink lung epithelial cells were grown overnight in 12-well clustereddishes in DMEM containing 10% fetal calf serum. The medium was thenchanged to DMEM containing 0.1% fetal calf serum and the cells wereincubated with 0.25 and 2.5 pM TGF-β₁ in the presence of variousconcentrations of β₁ ²⁵ (41-65) for 2.5 hr. Total cellular RNA wasextracted using RNAzol B (Tel-Test Inc.) according to the manufacturer'sprotocol. RNA was electrophoresed in 1.2% agarose-formaldehyde gel andtransferred to Duralon-UV membranes using 10×SCC. The membranes wereprobed at 42° C. with a random-primed, radiolabeled one kb fragment fromthe Hind III and NeoI digests of PAI-1 cDNA andglyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA. The blots werewashed with 0.1×SCC containing 0.1% SDS at room temperature.

References

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2. Laiho, M., Weis, F. M. B., and Massague, J. (1990) J. Biol. Chem.265:18518-18524.

3. Madison, L., Webb, N. R., Rose, T. M., Marquardt, H., Ikeda, T.,Twardzik, D., Seyedin, S., and Purchio, A. F. (1988) DNA and Cell Biol.7:18.

4. Schlunegger, M. P., and Grutter, M. G. (1992) Nature 353:430-434.

5. Hinck, A. P., Archer, S. J., Qian, S. W., Roberts, A. B., Sporn, M.B., Weatherbee, J. A., Tsang, M. L.-S., Lucas, R., Zhang, B.-L., Wenker,J., and Torchia, D. A. (1996) Biochem. 35:8517-8534.

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11 1 112 PRT Homo sapiens 1 Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser ThrGlu Lys Asn Cys Cys 1 5 10 15 Val Arg Gln Leu Tyr Ile Asp Phe Arg LysAsp Leu Gly Trp Lys Trp 20 25 30 Ile His Glu Pro Lys Gly Tyr His Ala AsnPhe Cys Leu Gly Pro Cys 35 40 45 Pro Tyr Ile Trp Ser Leu Asp Thr Gln TyrSer Lys Val Leu Ala Leu 50 55 60 Tyr Asn Gln His Asn Pro Gly Ala Ser AlaAla Pro Cys Cys Val Pro 65 70 75 80 Gln Ala Leu Glu Pro Leu Pro Ile ValTyr Tyr Val Gly Arg Lys Pro 85 90 95 Lys Val Glu Gln Leu Ser Asn Met IleVal Arg Ser Cys Lys Cys Ser 100 105 110 2 112 PRT Homo sapiens 2 Ala LeuAsp Ala Ala Tyr Cys Phe Arg Asn Val Gln Asp Asn Cys Cys 1 5 10 15 LeuArg Pro Leu Tyr Ile Asp Phe Lys Arg Asp Leu Gly Trp Lys Trp 20 25 30 IleHis Glu Pro Lys Gly Tyr Asn Ala Asn Phe Cys Ala Gly Ala Cys 35 40 45 ProTyr Leu Trp Ser Ser Asp Thr Gln His Ser Arg Val Leu Ser Leu 50 55 60 TyrAsn Thr Ile Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys Val Ser 65 70 75 80Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr Tyr Ile Gly Lys Thr Pro 85 90 95Lys Ile Glu Gln Leu Ser Asn Met Ile Val Lys Ser Cys Lys Cys Ser 100 105110 3 112 PRT Homo sapiens 3 Ala Leu Asp Thr Asn Tyr Cys Phe Arg Asn LeuGlu Glu Asn Cys Cys 1 5 10 15 Val Arg Pro Leu Tyr Ile Asp Phe Arg GlnAsp Leu Gly Trp Lys Trp 20 25 30 Val His Glu Pro Lys Gly Tyr Tyr Ala AsnPhe Cys Ser Gly Pro Cys 35 40 45 Pro Tyr Leu Arg Ser Ala Asp Thr Thr HisSer Thr Val Leu Gly Leu 50 55 60 Tyr Asn Thr Leu Asn Pro Glu Ala Ser AlaSer Pro Cys Cys Val Pro 65 70 75 80 Gln Asp Leu Glu Pro Leu Thr Ile LeuTyr Tyr Val Gly Arg Thr Pro 85 90 95 Lys Val Glu Gln Leu Ser Asn Met ValVal Lys Ser Cys Lys Cys Ser 100 105 110 4 25 PRT Homo sapiens 4 Ala AsnPhe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu Asp Thr 1 5 10 15 GlnTyr Ser Lys Val Leu Ala Leu Tyr 20 25 5 25 PRT Human 5 Ala Asn Phe CysAla Gly Ala Cys Pro Tyr Leu Trp Ser Ser Asp Thr 1 5 10 15 Gln His SerArg Val Leu Ser Leu Tyr 20 25 6 25 PRT Homo sapiens 6 Ala Asn Phe CysSer Gly Pro Cys Pro Tyr Leu Arg Ser Ala Asp Thr 1 5 10 15 Thr His SerThr Val Leu Gly Leu Tyr 20 25 7 10 PRT Homo sapiens 7 Pro Tyr Ile TrpSer Leu Asp Thr Gln Tyr 1 5 10 8 10 PRT Homo sapiens 8 Pro Tyr Leu TrpSer Ser Asp Thr Gln His 1 5 10 9 10 PRT Homo sapiens 9 Pro Tyr Leu ArgSer Ala Asp Thr Thr His 1 5 10 10 4 PRT Homo sapiens PEPTIDE (1)..(4)Residue 3 is any amino acid 10 Trp Ser Xaa Asp 1 11 4 PRT Homo sapiensPEPTIDE (1)..(4) Residue 3 is any amino acid 11 Arg Ser Xaa Asp 1

What I claim my invention is:
 1. A peptide of 10-25 amino acidscomprising amino acids 49-58 of a TGF-β₂, wherein the peptide is capableof inhibiting specific binding of a TGF-β to a TGF-β receptor on a cell.2. The peptide of claim 1, wherein the cell is a mink lung epithelialcell.
 3. The peptide of claim 1, comprising SEQ ID NO:8.
 4. The peptideof claim 3, consisting of SEQ ID NO:8.
 5. The peptide of claim 3,consisting of SEQ ID NO:5.
 6. A peptide of at least 10 amino acidscomprising amino acids 49-58 of a TGF-β₂, wherein the peptide is capableof blocking TGF-β-induced growth inhibition of a cell.
 7. The peptide ofclaim 6, wherein the cell is a mink lung epithelial cell.
 8. The peptideof claim 6, comprising SEQ ID NO:8.
 9. The peptide of claim 8,comprising SEQ ID NO:5.
 10. A peptide of at least 25 amino acidscomprising amino acids 41-65 of a TGF-β₃, wherein the peptide is capableof blocking TGF-β-induced growth inhibition of a cell.
 11. The peptideof claim 10, wherein the cell is a mink lung epithelial cell.
 12. Thepeptide of claim 10, comprising SEQ ID NO:6.
 13. A peptide of at least25 amino acids comprising amino acids 41-65 of a TGF-β₁, wherein thepeptide is capable of inhibiting specific binding of a TGF-β to a TGF-βreceptor on a cell.
 14. The peptide of claim 13, comprising SEQ ID NO:4.15. The peptide of claim 13, wherein the cell is a mink lung epithelialcell.
 16. A peptide of at least 25 amino acids comprising amino acids41-65 of a TGF-β₁, wherein the peptide is capable of blockingTGF-β-induced growth inhibition of a cell.
 17. The peptide of claim 16,comprising SEQ ID NO:4.
 18. The peptide of claim 16, wherein the cell isa mink lung epithelial cell.
 19. A method of inhibiting specific bindingof a TGF-β to a TGF-β receptor on a cell comprising contacting the cellwith a peptide of 10-25 amino acids, wherein (a) the peptide comprisesamino acids 49-58 of a TGF-β₁ or amino acids 49-58 of a TGF-β₂, and (b)the peptide inhibits the specific binding of a TGF-β to a TGF-β receptoron said cell.
 20. The method of claim 19, wherein the cell is a minklung epithelial cell.
 21. The method of claim 19, wherein the peptidecomprises SEQ ID NO:7 or SEQ ID NO:8.
 22. The method of claim 21,wherein the peptide comprises SEQ ID NO:7.
 23. The method of claim 22,wherein the peptide consists of SEQ ID NO:7.
 24. The method of claim 22,wherein the peptide consists of SEQ ID NO:4.
 25. The method of claim 21,wherein the peptide comprises SEQ ID NO:8.
 26. The peptide of claim 25,wherein the peptide consists of SEQ ID NO:8.
 27. The method of claim 25,wherein the peptide consists of SEQ ID NO:5.
 28. A method of blockingTGF-β-induced growth inhibition of a cell comprising contacting the cellwith a peptide of at least 10 amino acids, wherein (a) the peptidecomprises amino acids 49-58 of a TGF-β₂ or consists of amino acids 49-58of a TGF-β₁, and (b) the peptide blocks TGF-β-induced growth inhibitionof said cell.
 29. The method of claim 28, wherein the cell is a minklung epithelial cell.
 30. The method of claim 28, wherein the peptideconsists of SEQ ID NO:7.
 31. The method of claim 28, wherein the peptidecomprises SEQ ID NO:8.
 32. The method of claim 31, wherein the peptidecomprises SEQ ID NO:5.
 33. A method of blocking TGF-β-induced growthinhibition of a cell comprising contacting the cell with a peptide of atleast 25 amino acids, wherein (a) the peptide comprises amino acids41-65 of a TGF-β₁ or amino acids 41-65 of a TGF-β₃, and (b) the peptideblocks TGF-β-induced growth inhibition of said cell.
 34. The method ofclaim 33, wherein the cell is a mink lung epithelial cell.
 35. Themethod of claim 34, wherein the peptide comprises SEQ ID NO:4.
 36. Themethod of claim 33, wherein the peptide comprises SEQ ID NO:6.